Autophagy-based unconventional secretion in melanoma

Abstract

Macroautophagy, or autophagy, is a catabolic process that sequesters damaged organelles and protein aggregates for degradation. Autophagy occurs at basal levels in most cells to perform homeostatic functions and adaptively responds to environmental stresses to confer cell survival, serving a pro-tumorigenic role in established tumors. Autophagy has recently been recognized for its role in unconventional secretion, a novel paradigm beyond its previously known catabolic cellular function. However, its broader role in modifying the tumor secretome, a component of the tumor microenvironment that influences tumor progression and immune responses, has yet to be investigated. This is particularly relevant in melanoma, where autophagy levels are highly variable across tumors, considerably impacting tumor behavior and therapy response. In this thesis, we sought to determine the effects of autophagy at basal levels and in response to anti-melanoma therapy on the melanoma secretome to gain insight into the context-dependent effects of autophagy on secretion. The conditioned medium of isogenic melanoma cell lines with intrinsically disparate levels of autophagy were compared using quantitative proteomics, detecting 26 proteins at significantly higher levels in the secretomes of cells harboring high autophagic flux. From functional bioinformatics analysis, IL1-β, IL-8, LIF, DKK3, and FAM3C were the focus of further investigation. Measurements of these proteins in independent melanoma cell lines profiled for autophagy confirmed the early correlation with autophagy. Further, secretion of these proteins increased in response to targeted autophagy induction and decreased in autophagy-deficient melanoma cells. Finally, these proteins were detected at higher levels in sera of melanoma patients with high pre-treatment tumor autophagy levels, demonstrating the potential for improved clinical detection of autophagy. Follow up studies evaluated the effects of inhibiting BRAF, a serine-threonine kinase mutated in melanoma, on autophagy-based remodeling of the secretome. BRAF inhibition resulted in acute increases in autophagic flux in mutant BRAF cells coupled with increased secretion of a number of proteins, notably Histone H4 and ERK2 which were mechanistically linked to autophagy. Our work demonstrates that autophagy-based unconventional secretion is an important mechanism for altering the extracellular milieu in melanoma, the plasticity of which may impact tumor progression and therapy response in the clinic